Cécile Feuillie

Sticky Matrix: Adhesion Mechanism of the Staphylococcal Polysaccharide Intercellular Adhesin.

The development of bacterial biofilms on surfaces leads to hospital-acquired infections that are difficult to fight. In Staphylococci, the cationic polysaccharide intercellular adhesin (PIA) forms an extracellular matrix that connects the cells together during biofilm formation, but the molecular forces involved are unknown. Here, we use advanced force nanoscopy techniques to unravel the mechanism of PIA-mediated adhesion in a clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strain. Nanoscale multiparametric imaging of the structure, adhesion, and elasticity of bacteria expressing PIA shows that the cells are surrounded by a soft and adhesive matrix of extracellular polymers. Cell surface softness and adhesion are dramatically reduced in mutant cells deficient for the synthesis of PIA or under unfavorable growth conditions. Single-cell force spectroscopy demonstrates that PIA promotes cell-cell adhesion via the multivalent electrostatic interaction with polyanionic teichoic acids on the S. aureus cell surface. This binding mechanism rationalizes, at the nanoscale, the well-known ability of PIA to strengthen intercellular adhesion in staphylococcal biofilms. Force nanoscopy offers promising prospects for understanding the fundamental forces in antibiotic-resistant biofilms and for designing anti-adhesion compounds targeting matrix polymers.

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC

Significance The bacterial pathogen Staphylococcus aureus shows a remarkable ability to aggregate, thereby contributing to the formation of cellular communities that are difficult to eradicate. In this study, we dissect the homophilic interactions at play during S. aureus cell–cell adhesion, focusing on the key surface protein SdrC. We discover that SdrC is engaged in low-affinity homophilic bonds that promote intercellular adhesion, and that it also favors strong hydrophobic interactions with surfaces, emphasizing that this protein is a multifunctional adhesin. We also show that SdrC-dependent cell-surface attachment, cell–cell adhesion, and biofilm formation can be efficiently blocked by a peptide, thus suggesting this approach could be used for antibiofilm therapy. Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell–cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from β-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.

Forces between Staphylococcus aureus and human skin

Characterization of the molecular interactions between microbial cells and the human skin is essential to understand the functions of the skin microbiome, and to gain insight into the molecular basis of skin disorders. Although various molecular approaches have been used to study microbe-skin interactions, the underlying molecular forces were not accessible to study. Here we present a novel atomic force microscopy approach to localize and quantify the nanoscale interaction forces between the bacterial pathogen Staphylococcus aureus and human skin. A method combining nanoscale multiparametric imaging with single bacterial probes is developed to map simultaneously the topography and bacterial-binding properties of corneocytes at high spatiotemporal resolution. Further quantification of the forces between bacteria and corneocytes is achieved using single-cell force spectroscopy. The results show that the S. aureus-skin adhesion is strong (∼500 pN) and originates from multiple specific bonds between adhesins on the bacterial cell surface and target ligands on the corneocyte surface. Applicable to a wide variety of microbes and skin cells, our methodology offers exciting prospects for understanding the molecular details of skin colonization and infection.

Attachment of ribonucleotides on α-alumina as a function of pH, ionic strength, and surface loading.

The interactions between nucleic acids and mineral surfaces have been the focus of many studies in environmental sciences, in biomedicine, as well as in origin of life studies for the prebiotic formation of biopolymers. However, few studies have focused on a wide range of environmental conditions and the likely modes of attachment. Here we investigated the adsorption of ribonucleotides onto α-alumina surfaces over a wide range of pH, ionic strength, and ligand-to-solid ratio, by both an experimental and a theoretical approach. The adsorption of ribonucleotides is strongly affected by pH, with a maximum adsorption at pH values around 5. Alumina adsorbs high amounts of nucleotides >2 μmol/m(2). We used the extended triple-layer model (ETLM) to predict the speciation of the surface complexes formed as well as the stoichiometry and equilibrium constants. We propose the formation of two surface species: a monodentate inner-sphere complex, dominant at pH <7, and a bidentate outer-sphere complex, dominant at higher pH. Both complexes would involve interactions between the negatively charged phosphate group and the positively charged surface of alumina. Our results provide a better understanding of how nucleic acids attach to mineral surfaces under varying environmental conditions. Moreover, the predicted configuration of nucleotide surface species, bound via the phosphate group, could have implications for the abiotic formation of nucleic acids in the context of the origin of life.

Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis

ABSTRACT Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

Force-Induced Strengthening of the Interaction between Staphylococcus aureus Clumping Factor B and Loricrin.

ABSTRACT Bacterial pathogens that colonize host surfaces are subjected to physical stresses such as fluid flow and cell surface contacts. How bacteria respond to such mechanical cues is an important yet poorly understood issue. Staphylococcus aureus uses a repertoire of surface proteins to resist shear stress during the colonization of host tissues, but whether their adhesive functions can be modulated by physical forces is not known. Here, we show that the interaction of S. aureus clumping factor B (ClfB) with the squamous epithelial cell envelope protein loricrin is enhanced by mechanical force. We find that ClfB mediates S. aureus adhesion to loricrin through weak and strong molecular interactions both in a laboratory strain and in a clinical isolate. Strong forces (~1,500 pN), among the strongest measured for a receptor-ligand bond, are consistent with a high-affinity “dock, lock, and latch” binding mechanism involving dynamic conformational changes in the adhesin. Notably, we demonstrate that the strength of the ClfB-loricrin bond increases as mechanical force is applied. These findings favor a two-state model whereby bacterial adhesion to loricrin is enhanced through force-induced conformational changes in the ClfB molecule, from a weakly binding folded state to a strongly binding extended state. This force-sensitive mechanism may provide S. aureus with a means to finely tune its adhesive properties during the colonization of host surfaces, helping cells to attach firmly under high shear stress and to detach and spread under low shear stress. IMPORTANCE Staphylococcus aureus colonizes the human skin and the nose and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the S. aureus surface protein clumping factor B (ClfB) binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. Here, we unravel the molecular mechanism guiding the ClfB-loricrin interaction. We show that the ClfB-loricrin bond is remarkably strong, consistent with a high-affinity “dock, lock, and latch” binding mechanism. We discover that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of an S. aureus surface protein can be activated by physical stress. IMPORTANCE Staphylococcus aureus colonizes the human skin and the nose and can cause various disorders, including superficial skin lesions and invasive infections. During nasal colonization, the S. aureus surface protein clumping factor B (ClfB) binds to the squamous epithelial cell envelope protein loricrin, but the molecular interactions involved are poorly understood. Here, we unravel the molecular mechanism guiding the ClfB-loricrin interaction. We show that the ClfB-loricrin bond is remarkably strong, consistent with a high-affinity “dock, lock, and latch” binding mechanism. We discover that the ClfB-loricrin interaction is enhanced under tensile loading, thus providing evidence that the function of an S. aureus surface protein can be activated by physical stress.

Nucleation and growth of a bacterial functional amyloid at single-fiber resolution

Curli are functional amyloids produced by proteobacteria like Escherichia coli, as part of the extracellular matrix that holds cells together into biofilms. The molecular events during curli nucleation and fiber extension remain largely unknown. Combining observations from curli amyloidogenesis in bulk solutions with real-time in situ nanoscopic imaging at the single fiber level, we show that curli display polar growth, and detect two kinetic regimes of fiber elongation. Single fibers exhibit stop-and-go dynamics characterized by bursts of steady-state growth alternated with periods of stagnation. At high subunit concentrations fibers show constant, unperturbed burst growth. Curli follow a one-step nucleation process, where monomers contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to the mature fibrils. Kinetic data and interaction studies of curli fibrillation in the presence of the natural inhibitor CsgC show the inhibitor binds curli fibers and predominantly acts at the level of fiber elongation.

Binding of Nucleic Acid Components to the Serpentinite-Hosted Hydrothermal Mineral Brucite

The adsorption of nucleic acid components onto the serpentinite-hosted hydrothermal mineral brucite has been investigated experimentally by determining the equilibrium adsorption isotherms in aqueous solution. Thermodynamic characterization of the adsorption data has been performed using the extended triple-layer model (ETLM) to establish a model for the stoichiometry and equilibrium constants of surface complexes. Infrared characterization of the molecule-mineral complexes has helped gain insight into the molecular functional groups directly interacting with the mineral surface. Quantum mechanical calculations have been carried out to identify the possible complexes formed on surfaces by nucleic acid components and their binding configurations on mineral surfaces, both in the presence of water molecules and in water-free conditions. The results indicate that brucite favors adsorption of nucleotides with respect to nucleosides and nucleobases from dilute aqueous environments. The surface of this mineral is able to induce well-defined orientations of the molecules through specific molecule-mineral interactions. This result suggests plausible roles of the mineral brucite in assisting prebiotic molecular self-organization. Furthermore, the detection of the infrared spectroscopic features of such building blocks of life adsorbed on brucite at very low degrees of coverage provides important support to life detection investigations.

Forces guiding staphylococcal adhesion.

Staphylococcus epidermidis and Staphylococcus aureus are two important nosocomial pathogens that form biofilms on indwelling medical devices. Biofilm infections are difficult to fight as cells within the biofilm show increased resistance to antibiotics. Our understanding of the molecular interactions driving bacterial adhesion, the first stage of biofilm formation, has long been hampered by the paucity of appropriate force-measuring techniques. In this minireview, we discuss how atomic force microscopy techniques have enabled to shed light on the molecular forces at play during staphylococcal adhesion. Specific highlights include the study of the binding mechanisms of adhesion molecules by means of single-molecule force spectroscopy, the measurement of the forces involved in whole cell interactions using single-cell force spectroscopy, and the probing of the nanobiophysical properties of living bacteria via multiparametric imaging. Collectively, these findings emphasize the notion that force and function are tightly connected in staphylococcal adhesion.

Bacterial Sexuality at the Nanoscale

Understanding the basic mechanisms of bacterial sexuality is an important topic in current microbiology and biotechnology. While classical methods used to study gene transfer provide information on whole cell populations, nanotechnologies offer new opportunities for analyzing the behavior of individual mating partners. We introduce an innovative atomic force microscopy (AFM) platform to study and mechanically control DNA transfer between single bacteria, focusing on the large conjugative pXO16 plasmid of the Gram-positive bacterium Bacillus thuringiensis. We demonstrate that the adhesion forces between single donor and recipient cells are very strong (∼2 nN). Using a mutant plasmid, we find that these high forces are mediated by a pXO16 aggregation locus that contains two large surface protein genes. Notably, we also show that AFM can be used to mechanically induce plasmid transfer between single partners, revealing that transfer is very fast (<15 min) and triggers major cell surface changes in transconjugant cells. We anticipate that the single-cell technology developed here will enable researchers to mechanically control gene transfer among a wide range of Gram-positive and Gram-negative bacterial species and to understand the molecular forces involved. Also, the method could be useful in nanomedicine for the design of antiadhesion compounds capable of preventing intimate cell-cell contacts, therefore providing a means to control the resistance and virulence of bacterial pathogens.